ABSTRACT Emerin is an integral membrane protein of the inner nuclear envelope (INE) that binds to various nucleoskeletal partners and is a critical actor for the maintenance of the nucleus architecture and for nuclear mechanotransductions in response to forces. When mutated or absent, emerin causes Emery-Dreifuss muscular dystrophy (EDMD), an envelopathy whose underlying mechanisms and muscle specific effects are not fully understood. In particular, how emerin participates in molecular scaffolding at the INE and helps protect the nucleus against mechanical strains has remained largely elusive. As we have shown recently, using state-of-the-art optical microscopy, this is because the spatial organization of emerin and its mechanotransducing functions are modulated on distances of just a few nanometers at the INE, a length-scale inaccessible by conventional microscopy imaging techniques. Defining the pathogenesis of EDMD and other envelopathies, therefore demand new approaches that can establish the nanoscale structural organization of the INE while simultaneously modulating the mechanical landscape of nuclei in intact cells. Here, we propose an innovative integration of super-resolution and single molecule optical microscopy, nuclear biomechanics, biochemistry and quantitative biophysical analyses: (i) to establish the structural organization and the mechanotransducing functions of emerin at the nanoscale in human cells and (ii) to uncover the mechanisms by which its mutation results in abnormal nuclear mechanics in EDMD. Building on a large set of preliminary data with mutated emerins that localize correctly to the NE but induce EDMD, we hypothesize that the nanoscale oligomerization of emerin is directly coupled to its mechanotransducing functions and is further regulated by competitive interactions with various nucleoskeletal niches. We also hypothesize that defective nuclear biomechanics stem from dysregulated emerin oligomerization and, consequently, defective organization of structural niches at the INE. These hypotheses will be tested in two aims. In Aim 1, we will determine how nucleoskeletal niches differentially enriched in key emerin binding partners modulate the diffusion dynamics and the oligomeric state of wild-type emerin and a variety of EDMD-inducing emerin mutants at the INE of rescued emerin-null cells from human EDMD patients. This will be done using multi-parametric super-resolution microscopy, single molecule tracking and biochemical assays. In Aim 2, we will define the functional roles of emerin as a key nuclear envelope mechanotransducer in the context of EDMD by establishing how changes in the nanoscale distribution and oligomerization of wild-type emerin or emerin mutants dictate the normal or pathogenic mechanical responses of cell nuclei to increasing forces. This will be achieved using cell micropatterning approaches that controllably modulate the mechanical landscape of nuclei directly in cells. Together, this research project will greatly advance our fundamental understanding of mechanotransduction processes at the NE of human cells and will provide a completely new outlook on the function of emerin and the pathogenesis of EDMD. This work will also have a tangible impact on the vast array of nuclear envelope disorders by significantly improving our understanding of the diseases and by providing novel targets to develop therapies for patients affected by these pathologies.